Activation of the cJun-N-terminal kinase (JNK) map kinase (MAPK) module plays a major role in regulating responses to cellular stress, inflammatory cytokines and normal developmental processes. We have previously identified a mammalian Ste20 kinase NIK (Nck-Interacting Kinase) that specifically activates the JNK MAPK module. A Drosophila homolog of NIK, misshapen (msn) has recently been identified, and we found that msn mutant Drosophila die embryonically with a dorsal open phenotype due to failure to activate the JNK MAPK module. Our central hypothesis is that msn, and NIK are components of a signaling pathway that is conserved between flies and mammals to control developmentally regulated pathways via the activation of the JNK MAPK module. The major goal of this research proposal is to elucidate the regulation and function of mammalian NIK and its Drosophila homolog msn. We plan: (1) To identify the domains of msn and NIK that are responsible for JNK activation in a biologically relevant system. We will perform structure/function studies to determine the domains of msn and NIK that mediate JNK activation using an in vivo model in Drosophila. (a) We will test whether msn requires an intact kinase domain, C-terminal regulatory domain and a third region that has been shown to mediate the association with TNF Receptor Associated Factors (TRAF) to couple to JNK activation. (b) we will determine whether Ste20 kinases related to NIK rescue the dorsal open phenotype in msn -/- embryos. (2) We will test the hypothesis that the Drosophila SH2/SH3 adaptor dreadlock (dock) functions in R-cell targeting as an adaptor molecule, coupling msn bound to its SH3 domain to a receptor tyrosine kinase localized to the axonal growth cone. To assess whether msn interacts genetically with dock to mediate photoreceptor axonal targeting, we will determine whether msn mutant R cells have a defect in axonal targeting that is similar to dock mutants, determine genetically whether msn and dock function on the same pathway, and determine the order in which they function. (3) To identify the upstream signals and downstream targets of Msn and NIK. Using the yeast-two hybrid system, we found that msn binds a novel Drosophila TRAF. We will test whether TRAFs function as adaptor molecules to couple msn in Drosophila and NIK in mammalian cells to specific upstream signals that regulate JNK. (4) To determine the role of NIK in mammalian development by creating a mouse nullizygous for NIK. NIK minus cell lines will be derived from these mice, and these cell lines will be used to determine which upstream signals are dependent upon NIK to activate the JNK MAP kinase pathway.